Ultrastructural relationships between cortical,thalamic, and amygdala glutamatergic inputs and group I metabotropic glutamate receptors in the rat accumbens

Changes in glutamatergic transmission in the nucleus accumbens play a key role in mediating reward‐related behaviors and addiction to psychostimulants. Glutamatergic inputs to the accumbens originate from multiple sources, including the prefrontal cortex, basolateral amygdala, and midline thalamus. The group I metabotropic glutamate receptors (mGluRs) are found throughout the core and shell of the nucleus accumbens, but their localization and function at specific glutamatergic synapses remain unknown. To further characterize the substrate that underlies group I mGluR functions in the accumbens, we combined anterograde tract tracing method with electron microscopy immunocytochemistry to study the ultrastructural relationships between specific glutamatergic afferents and mGluR1a‐ or mGluR5‐containing neurons in the rat nucleus accumbens. Although cortical, thalamic, and amygdala glutamatergic terminals contact both mGluR1a‐ and mGluR5‐immunoreactive dendrites and spines in the shell and core of the accumbens, they do so to varying degrees. Overall, glutamatergic terminals contact mGluR1a‐positive spines about 30% of the time, whereas they form synapses twice as frequently with mGluR5‐labeled spines. At the subsynaptic level, mGluR5 is more frequently expressed perisynaptically and closer to the edges of glutamatergic axospinous synapses than mGluR1a, suggesting a differential degree of activation of the two group I mGluRs by transmitter spillover from glutamatergic synapses in the rat accumbens. These results lay the foundation for a deeper understanding of group I mGluR‐mediated effects in the ventral striatum, and their potential therapeutic benefits in drug addiction and other neuropsychiatric changes in reward‐related behaviors. J. Comp. Neurol. 518:1315–1329, 2010. © 2009 Wiley‐Liss, Inc.     

Distribution of mGluR1alpha and mGluR5 immunolabeling in primate prefrontal cortex

Metabotropic glutamate receptors (mGluRs) mediate important modulatory glutamatergic influences throughout the brain. However, the specific localization and functions of group I mGluR subtypes (mGluR1alpha and mGluR5) in cortical neurotransmission are not well known, particularly in primates. To address this issue, we used immunoelectron microscopy to compare the subcellular localizations of mGluR1alpha and mGluR5 in the prefrontal cortex of macaque monkeys. Both receptor subtypes were found in a variety of subcellular compartments, including spines, dendrites, preterminal axons, axon terminals, and glia; however, quantitative differences were found in the relative abundance of labeled elements for each receptor. The mGluR1alpha-immunoreactive (-IR) elements were overwhelmingly the spines and dendrites, with labeled terminals, axons, and glia seen more rarely. The mGluR5-IR elements were also mostly spines and dendrites, but the proportion of labeled unmyelinated axons, terminals, and glia was higher than for mGluR1alpha-IR elements. Double labeling with SMI-32 and parvalbumin confirmed that both receptors were found in pyramidal cell and interneuron dendrites. The localization of mGluR1alpha to pyramidal cells in primate cortex contrasts with reports that mGluR1alpha is found almost exclusively in interneurons in rodent cortex. By using double labeling, we found no evidence for mGluR1alpha or mGluR5 in dopaminergic afferents to prefrontal cortex. The data presented here provide an anatomical substrate for a differential role of mGluR1alpha and mGluR5 in post-and presynaptic actions of glutamate in primate prefrontal cortex. They further suggest differences in the cortical distribution of group I mGluRs between primates and rodents.     

Subcellular and subsynaptic localization of group I metabotropic glutamate receptors in the monkey subthalamic nucleus

Both subtypes of group I metabotropic glutamate receptor, mGluR1 and mGluR5, are expressed postsynaptically in neurons of the subthalamic nucleus (STN), and their activation induces different physiological responses. To test whether these effects could be explained by a differential localization of the two group I mGluRs, we analyzed the subcellular and subsynaptic distribution of mGluR1a and mGluR5 in the monkey STN. Double-immunofluorescence and light microscopic analyses revealed that both group I mGluR subtypes were strongly coexpressed in the neuropil and neuronal perikarya. Astrocytic perikarya exhibited intense mGluR1a, but no detectable mGluR5, immunoreactivity. At the electron microscopic level, immunoperoxidase labeling for both mGluR1a and mGluR5 was localized mainly in dendrites. A significant proportion of the total pool of mGluR1a-immunoreactive elements was accounted for by glial cell processes, whereas glial cell labeling was much less frequently encountered in sections immunostained for mGluR5. Preembedding immunogold labeling in STN dendrites revealed that 60-70% of the gold labeling for both mGluR subtypes was intracellular, whereas 30-40% was apposed to the plasma membrane. Of the plasma membrane-apposed particles, more than 90% were extrasynaptic; fewer than 10% were associated with symmetric or asymmetric synapses. Most of the synapse-associated labeling was found at the edges of both asymmetric and symmetric postsynaptic specializations. Some extrasynaptic gold particles were aggregated on parts of the plasma membrane tightly apposed by glial processes. These findings demonstrate that mGluR1a and mGluR5 exhibit a similar pattern of subsynaptic localization in monkey STN neurons, with both receptor subtypes exhibiting substantial extrasynaptic and perisynaptic localization.     

Metabotropic glutamate receptors in superficial laminae of the rat dorsal horn.

Light and electron microscopic immunocytochemistry were employed here to show the distribution of metabotropic glutamate receptors (mGluRs) mGluR2/3 and mGluR5 in laminae I and II of the dorsal horn, to identify their pre- and postsynaptic location, and to test colocalization with gamma-aminobutyric acid (GABA). mGluR2/3 was mainly in the inner part of lamina II; mGluR5 was mainly in laminae I and II. Electron microscopy showed that both mGluR2/3 and mGluR5 were in perikarya, dendrites, and vesicle-containing profiles. Two main morphological types of primary afferent terminals can be distinguished in the superficial laminae: C1, likely to be endings of unmyelinated fibers, and C2, of small myelinated fibers. Quantitative data show that only a small fraction of C2s stained for either receptor; more common were immunopositive dendrites postsynaptic to these terminals, and most common were appositions between C2s and mGluR5 immunopositive dendrites. Vesicle-containing profiles were characteristically apposed to primary afferent terminals, mainly C2s. Immunopositivity for mGluRs, especially mGluR2/3, was present in vesicle-containing profiles apposed to C2, none to C1, and about half of the profiles immunostained for either receptor were also stained for GABA. The presence of presynaptic and postsynaptic mGluRs in both inhibitory and excitatory interneurons may contribute to complex processing of fast and slow responses to peripheral input in superficial laminae. As selective agonists of mGluRs may modulate GABA release, the present demonstration of mGluRs in GABAergic terminals of presumed interneurons suggests that facilitatory effects may involve a mechanism of disinhibition.     

Age-related changes in the expression of axonal and glial group I metabotropic glutamate receptor in the rat substantia nigra pars reticulata

Neuronal systems undergo many significant changes during the course of brain development. To characterize the developmental changes in the substantia nigra pars reticulata (SNr) associated with the expression of group I metabotropic glutamate receptors (mGluRs), we used the immunoperoxidase and immunogold methods at the electron microscope level to determine whether the subcellular and subsynaptic patterns of distribution of mGluR1a and mGluR5 differ between young (P14-P18) and adult (>2 months) rats. The SNr of young rats contained a significantly higher density of labeled unmyelinated axons for both receptor subtypes. In addition, mGluR5-immunoreactive glial processes were very abundant in young rats but absent in the adults. On the other hand, the relative proportion of immunoreactive dendrites was the same for both age groups. Analysis of immunogold-labeled rat SNr revealed similar proportions of plasma membrane-bound mGluR1a and mGluR5 in adult (59.8 and 19.4%, respectively) and young (60.6 and 18.4%, respectively) rats. The pattern of subsynaptic localization of mGluR1a also remained the same between young and adults. However, the proportion of extrasynaptic mGluR5 decreased, whereas proportions of gold particles associated with symmetric synapses increased in adults. The results of this study demonstrate significant differences in the expression of group I mGluRs in the SNr of young and adult rats. These findings support a role for group I mGluRs during development and emphasize the importance of using brain tissue from age-matched subjects when attempting to correlate functional data from young rat brain slices with immunocytochemical localization of group I mGluRs.     

Functional and ultrastructural analysis of group I mGluR in striatal fast-spiking interneurons

Striatal parvalbumin-containing fast-spiking (FS) interneurons provide a powerful feedforward GABAergic inhibition on spiny projection neurons, through a widespread arborization and electrical coupling. Modulation of FS interneuron activity might therefore strongly affect striatal output. Metabotropic glutamate receptors (mGluRs) exert a modulatory action at various levels in the striatum. We performed electrophysiological recordings from a rat striatal slice preparation to investigate the effects of group I mGluR activation on both the intrinsic and synaptic properties of FS interneurons. Bath-application of the group I mGluR agonist, (S)-3,5-dihydroxyphenylglycine (3,5-DHPG), caused a dose-dependent depolarizing response. Both (S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385) and 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), selective mGluR1 antagonists, significantly reduced the amplitude of the membrane depolarization caused by 3,5-DHPG application. Conversely, mGluR5 antagonists, 2-methyl-6-(phenylethylnyl)pyridine hydrochloride (MPEP) and 6-methyl-2-(phenylazo)-3-pyridinol (SIB1757), were unable to affect the response to 3,5-DHPG, suggesting that only mGluR1 contributes to the 3,5-DHPG-mediated excitatory action on FS interneurons. Furthermore, mGluR1 blockade significantly decreased the amplitude of the glutamatergic postsynaptic potentials, whereas the mGluR5 antagonist application produced a small nonsignificant inhibitory effect. Surprisingly, our electron microscopic data demonstrate that the immunoreactivity for both mGluR1a and mGluR5 is expressed extrasynaptically on the plasma membrane of parvalbumin-immunoreactive dendrites of FS interneurons. Together, these results suggest that despite a common pattern of distribution, mGluR1 and mGluR5 exert distinct functions in the modulation of FS interneuron activity.     

Group I metabotropic glutamate receptors at GABAergic synapses in monkeys.

Recent data showed that group I metabotropic glutamate receptors (mGluRs) are located perisynaptic to the postsynaptic specializations of asymmetric glutamatergic synapses in the cerebellum and hippocampus in rats. In the present study, we used immunogold labeling to elucidate the subsynaptic localization of group I mGluRs (mGluR1a and mGluR5) in the internal and external segments of the globus pallidus in monkeys. In contrast to hippocampal and cerebellar neurons, which receive massive glutamatergic inputs, dendrites of pallidal neurons are covered with GABAergic boutons from the striatum intermingled with a small proportion of glutamatergic terminals arising largely from the subthalamic nucleus. In line with previous data, mGluR1a and mGluR5 immunoreactivity was found at the edge of the postsynaptic specializations of asymmetric synapses established by subthalamic-like boutons in the monkey pallidum. However, a large proportion of gold particles were also seen in the main body of the postsynaptic specializations of symmetric synapses formed by striatal GABAergic terminals. These data raise questions about the possible sources of activation of these receptors and the potential roles of group I mGluRs in modulating GABAergic neurotransmission at striatopallidal synapses.     

High level of mGluR7 in the presynaptic active zones of select populations of GABAergic terminals innervating interneurons in the rat hippocampus

The release of neurotransmitters is modulated by presynaptic metabotropic glutamate receptors (mGluRs), which show a highly selective expression and subcellular location in glutamatergic terminals in the hippocampus. Using immunocytochemistry, we investigated whether one of the receptors, mGluR7, whose level of expression is governed by the postsynaptic target, was present in GABAergic terminals and whether such terminals targeted particular cells. A total of 165 interneuron dendritic profiles receiving 466 synapses (82% mGluR7a-positive) were analysed. The presynaptic active zones of most GAD-(77%) or GABA-positive (94%) synaptic boutons on interneurons innervated by mGluR7a-enriched glutamatergic terminals (mGluR7a-decorated) were immunopositive for mGluR7a. GABAergic terminals on pyramidal cells and most other interneurons in str. oriens were mGluR7a-immunonegative. The mGluR7a-decorated cells were mostly somatostatin- and mGluR1alpha-immunopositive neurons in str. oriens and the alveus. Their GABAergic input mainly originated from VIP-positive terminals, 90% of which expressed high levels of mGluR7a in the presynaptic active zone. Parvalbumin-positive synaptic terminals were rare on mGluR7a-decorated cells, but on these neurons 73% of them were mGluR7a-immunopositive. Some type II synapses innervating interneurons were immunopositive for mGluR7b, as were some type I synapses. Because not all target cells of VIP-positive neurons are known it has not been possible to determine whether mGluR7 is expressed in a target-cell-specific manner in the terminals of single GABAergic cells. The activation of mGluR7 may decrease GABA release to mGluR7-decorated cells at times of high pyramidal cell activity, which elevates extracellular glutamate levels. Alternatively, the presynaptic receptor may be activated by as yet unidentified endogenous ligands released by the GABAergic terminals or the postsynaptic dendrites.     

Depression of GABAergic input to identified hippocampal neurons by group III metabotropic glutamate receptors in the rat

The release of GABA in synapses is modulated by presynaptic metabotropic glutamate receptors (mGluRs). We tested whether GABA release to identified hippocampal neurons is influenced by group III mGluR activation using the agonist L-(+)-2-amino-4-phosphonobutyric acid (L-AP4) on inhibitory postsynaptic currents (IPSCs) evoked in CA1 interneurons and pyramidal cells. In interneurons, characterized with biocytin and immunolabelling for somatostatin, evoked IPSCs were depressed by 50 micro m L-AP4 (activating mGluR4 and 8) to 68 +/- 6% of control, but they were rarely depressed in pyramidal cells (96 +/- 4% of control). At 300-500 micro m concentration (activating mGluR4, 7 and 8), L-AP4 depressed IPSCs in both interneurons (to 70 +/- 6%) and pyramidal cells (to 67 +/- 4%). The change in trial-to-trial variability and in paired-pulse depression indicated a presynaptic action. In interneurons, the degree of IPSC depression was variable (to 9-87%), and a third of IPSCs were not affected by L-AP4. The L-AP4-evoked IPSC depression was blocked by LY341495. The depression of IPSCs was similar in O-LM cells and other interneurons. The lack of cell-type selectivity and the similar efficacy of different concentrations of L-AP4 suggest that several group III mGluRs are involved in the depression of IPSCs. Electron microscopic immunocytochemistry confirmed that mGluR4, mGluR7a and mGluR8a occur in the presynaptic active zone of GABAergic terminals on interneurons, but not on those innervating pyramidal cells. The high variability of L-AP4-evoked IPSC suppression is in line with the selective expression of presynaptic mGluRs by several distinct types of GABAergic neuron innervating each interneuron type.     

Differential effects of the group II mGluR agonist, DCG-IV, on depolarization-induced suppression of inhibition in hippocampal CA1 and CA3 neurons

We investigated the role of metabotropic glutamate receptors in the mediation of depolarization-induced suppression of inhibition (DSI), using whole-cell electrophysiological techniques in rat hippocampal slice preparation. In a previous work, we showed that a retrograde signal travels from CA1 pyramidal cells to GABA interneurons and prevents them from releasing GABA for tens of seconds at 30 degrees C. The resulting suppression of inhibition is DSI. The retrograde signal appeared to be glutamate, or a glutamate analog, which acted on group I metabotropic receptors on the interneurons. It is not known if DSI occurs in hippocampal subregions besides CA1. If DSI does occur in other regions, it will be important to know if the role of metabotropic glutamate receptors (mGluRs) in mediating DSI is the same everywhere. The distribution of mGluR subtypes varies among hippocampal subregions. In the CA3 region, unlike CA1, group II mGluRs are prevalent. It was possible, therefore, that in CA3, the group II mGluRs would mediate DSI. We have begun to investigate these issues. We now report that: 1) DSI does occur in CA3. 2) Carbachol induces IPSC activity that can be recorded in CA1 and CA3a. This carbachol-induced activity can be reduced by the selective group II mGluR agonist, DCG-IV, and by DSI. 3) Evoked IPSCs in CA3a, but not in CA1, can be reduced by DCG-IV; hence the interneurons activated by carbachol may reside in CA3a. 4) Despite the group II mGluR agonist sensitivity of CA3a interneurons, DSI in this region is not affected by a group II mGluR antagonist, CPPG, and therefore does not appear to be mediated by group II mGluRs.     

Subregional, dendritic compartment, and spine subtype specificity in cocaine regulation of dendritic spines in the nucleus accumbens

Numerous studies have found that chronic cocaine increases dendritic spine density of medium spiny neurons in the nucleus accumbens (NAc). Here, we used single-cell microinjections and advanced 3D imaging and analysis techniques to extend these findings in several important ways: by assessing cocaine regulation of dendritic spines in the core versus shell subregions of NAc in the mouse, over a broad time course (4 h, 24 h, or 28 d) of withdrawal from chronic cocaine, and with a particular focus on proximal versus distal dendrites. Our data demonstrate subregion-specific, and in some cases opposite, regulation of spines by cocaine on proximal but not distal dendrites. Notably, all observed density changes were attributable to selective regulation of thin spines. At 4 h after injection, the proximal spine density is unchanged in the core but significantly increased in the shell. At 24 h, the density of proximal dendritic spines is reduced in the core but increased in the shell. Such downregulation of thin spines in the core persists through 28 d of withdrawal, whereas the spine density in the shell returns to baseline levels. Consistent with previous results, dendritic tips exhibited upregulation of dendritic spines after 24 h of withdrawal, an effect localized to the shell. The divergence in regulation of proximal spine density in NAc core versus shell by cocaine correlates with recently reported electrophysiological data from a similar drug administration regimen and might represent a key mediator of changes in the reward circuit that drive aspects of addiction.     

Immunolocalization of mGluR1α in specific populations of local circuit neurons in the cerebral cortex

By coupling glutamate to the IP₃ signaling pathway, group I metabotropic receptors can increase intracellular Ca²⁺ concentration, and might thus contribute to excitotoxicity. To identify neurons that might be vulnerable to such injury, we performed immunofluorescence histochemistry for metabotropic glutamate receptor 1α (mGluR1α) in the cerebral cortex of adult rat. mGluR1α was in somata and dendrites of a subset of non-pyramidal neurons scattered throughout the cerebral cortex. To further characterize mGluR1α-positive neurons, we investigated its colocalization with several neurochemical markers. Nearly all mGluR1α-positive cells were interneurons immunopositive for γ-aminobutyric acid. The majority (70–80%) of mGluR1α-immunopositive neurons were double-labeled for somatostatin. Approximately half of calretinin-positive neurons and 30% of calbindin-positive neurons expressed mGluR1α. In contrast, parvalbumin-expressing neurons were rarely positive for mGluR1α. Neurons staining strongly for mGluR1α were also positive for GluR1. These results indicated that mGluR1α is expressed by specific classes of GABAergic neurons in the neocortex, and suggests a mechanism by which these neurons may be especially vulnerable to excitotoxic injury.     

Neurokinin 1 receptor distribution in cholinergic neurons and targets of substance P terminals in the rat nucleus accumbens

Substance P (SP) is the major endogenous ligand for neurokinin 1 (NK1) receptors and, together with acetylcholine, has an important role in motivated behaviors involving the limbic shell and motor core of the nucleus accumbens (NAc). To determine the functional sites for SP activation of NK-1 receptors and potential interactions with cholinergic neurons in these regions, the authors examined the electron microscopic immunocytochemical localization either of antisera against the NK1 receptor or of the NK1 receptor and either 1) SP or 2) the vesicular acetylcholine transporter (VAchT) in rat NAc. In both the NAc shell and core, NK1 receptor labeling was localized mainly to somatic and dendritic plasma membranes and nearby endosomal organelles in aspiny neurons. In sections through the ventromedial shell that were processed for NK1/SP labeling, 46% of the NK1-immunoreactive dendrites (n = 603 dendrites) showed symmetric or appositional contacts with SP-containing terminals. These terminals and several others that formed symmetric synapses also occasionally were immunoreactive for NK1 receptors. Analysis of the shell region for NK1/VAchT labeling showed that 61% of the total immunoreactive dendrites (n = 534 dendrites) contained NK1 receptors without VAchT, 29% contained both products, and 10% contained VAchT only. Many of the labeled somata and dendrites also received synaptic contact from VAchT-containing terminals. These findings suggest that, in the NAc, NK1 receptors are recycled through endosomal compartments and play a role in modulating mainly the postsynaptic responses, but also the presynaptic release, of SP and/or inhibitory neurotransmitters onto aspiny interneurons, some of which are cholinergic.     

Adenosine A2A receptor in the monkey basal ganglia: ultrastructural localization and colocalization with the metabotropic glutamate receptor 5 in the striatum

The adenosine A(2A) receptor (A(2A) R) is a potential drug target for the treatment of Parkinson's disease and other neurological disorders. In rodents, the therapeutic efficacy of A(2A) R modulation is improved by concomitant modulation of the metabotropic glutamate receptor 5 (mGluR5). To elucidate the anatomical substrate(s) through which these therapeutic benefits could be mediated, pre-embedding electron microscopy immunohistochemistry was used to conduct a detailed, quantitative ultrastructural analysis of A(2A) R localization in the primate basal ganglia and to assess the degree of A(2A) R/mGluR5 colocalization in the striatum. A(2A) R immunoreactivity was found at the highest levels in the striatum and external globus pallidus (GPe). However, the monkey, but not the rat, substantia nigra pars reticulata (SNr) also harbored a significant level of neuropil A(2A) R immunoreactivity. At the electron microscopic level, striatal A(2A) R labeling was most commonly localized in postsynaptic elements (58% ± 3% of labeled elements), whereas, in the GPe and SNr, the labeling was mainly presynaptic (71% ± 5%) or glial (27% ± 6%). In both striatal and pallidal structures, putative inhibitory and excitatory terminals displayed A(2A) R immunoreactivity. Striatal A(2A) R/mGluR5 colocalization was commonly found; 60-70% of A(2A) R-immunoreactive dendrites or spines in the monkey striatum coexpress mGluR5. These findings provide the first detailed account of the ultrastructural localization of A(2A) R in the primate basal ganglia and demonstrate that A(2A) R and mGluR5 are located to interact functionally in dendrites and spines of striatal neurons. Together, these data foster a deeper understanding of the substrates through which A(2A) R could regulate primate basal ganglia function and potentially mediate its therapeutic effects in parkinsonism.     

Perisynaptic Location of Metabotropic Glutamate Receptors mGluR1 and mGluR5 on Dendrites and Dendritic Spines in the Rat Hippocampus

Ionotropic and metabotropic (mGluR1a) glutamate receptors were reported to be segregated from each other within the postsynaptic membrane at individual synapses. In order to establish whether this pattern of distribution applies to the hippocampal principal cells and to other postsynaptic metabotropic glutamate receptors, the mGluR1a/b/c and mGluR5 subtypes were localized by immunocytochemistry. Principal cells in all hippocampal fields were reactive for mGluR5, the strata oriens and radiatum of the CA1 area being most strongly immunolabelled. Labelling for mGluR1b/c was strongest on some pyramids in the CA3 area, weaker on granule cells and absent on CA1 pyramids. Subpopulations of non-principal cells showed strong mGluR1 or mGluR5 immunoreactivity. Electron microscopic pre-embedding immunoperoxidase and both pre- and postembedding immunogold methods consistently revealed the extrasynaptic location of both mGluRs in the somatic and dendritic membrane of pyramidal and granule cells. The density of immunolabelling was highest on dendritic spines. At synapses, immunoparticles for both mGluR1 and mGluR5 were found always outside the postsynaptic membrane specializations. Receptors were particularly concentrated in a perisynaptic annulus around type I synaptic junctions, including the invaginations at 'perforated'synapses. Measurements of immunolabelling on dendritic spines showed decreasing levels of receptor as a function of distance from the edge of the synaptic specialization. We propose that glutamatergic synapses with an irregular edge develop in order to increase the circumference of synaptic junctions leading to an increase in the metabotropic to ionotropic glutamate receptor ratio at glutamate release sites. The perisynaptic position of postsynaptic metabotropic glutamate receptors appears to be a general feature of glutamatergic synaptic organization and may apply to other G-protein-coupled receptors.     

Differentially altered mGluR1 and mGluR5 mRNA expression in rat caudate nucleus and nucleus accumbens in the development and expression of behavioral sensitization to repeated amphetamine administration.

Altered glutamatergic transmission in the striatum may be implicated in behavioral sensitization to repeated amphetamine (AMPH) administration. Quantitative in situ hybridization histochemistry was performed to define the effects of acute and chronic AMPH exposures on mRNA expression of Group I metabotropic glutamate receptors (mGluRs) in the striatum. Behavioral ratings indicated that the motor activity of rats was significantly higher after the final of five daily AMPH injections (4 mg/kg, i.p.) than that after the first of five daily AMPH, indicative of the development of behavioral sensitization. Moreover, the motor activity of rats treated with five daily AMPH was significantly greater than that of rats treated with five daily saline in response to a 2 mg/kg challenge dose of AMPH 7, 14, 28, and 60 days after the discontinuation of drug treatments, indicative of the persistent expression of behavioral sensitization. Three hours after acute administration of AMPH to naive rats, mGluR1 and mGluR5 mRNA expression in the dorsal (caudatoputamen) and ventral (nucleus accumbens) striatum showed no change as compared to acute saline injection. In rats that developed behavioral sensitization to repeated AMPH, mGluR1 levels in the dorsal and ventral striatum were increased by 53% and 43%, respectively, 3 h after the final AMPH treatment. However, this change did not persist during withdrawal since it was not observed 7, 14, and 28 days after the discontinuation of AMPH treatment. Conversely, mGluR5 levels were markedly reduced 3 h after the final of five daily AMPH treatments in the entire striatum of sensitized rats (34% and 77% of controls in the dorsal and ventral striatum, respectively). The reduction persisted at 7, 14, and 28 days of withdrawal. These results reveal a close linkage between striatal Group I mGluR gene expression and behavioral sensitization to AMPH. This may indicate functional implications of the two subtypes of Group I mGluRs in the regulation of behavioral sensitization to the dopamine stimulant.     

Immunolocalization of metabotropic glutamate receptor 1alpha (mGluR1alpha) in distinct classes of interneuron in the CA1 region of the rat hippocampus

In the hippocampal CA1 region, metabotropic glutamate subtype 1 (mGluR1) receptors have been implicated in a variety of physiological responses to glutamate, which include modulation of synaptic transmission and plasticity, as well as neuronal excitability and synchronization. The mGluR1alpha isoform is characteristically expressed only by nonprincipal cells, and it is particularly enriched in somatostatin (SS)-containing interneurons in stratum oriensalveus. Anatomical and physiological data have indicated the presence of mGluR1alpha in several distinct classes of interneurons with their somata located also in strata pyramidale, radiatum, and lacunosum moleculare. Each different interneuron subtype, as defined by functionally relevant criteria, including input/ output characteristics and expression of selective molecular markers, subserves distinct functions in local hippocampal circuits. We have investigated which of the different CA1 interneuron classes express mGluR1alpha by immunofluorescent labeling, combining antibodies to mGluR1alpha, calcium-binding proteins, and neuropeptides, and by intracellular labeling in vitro. Several types of interneuron that are immunopositive for mGluR1alpha each targeted different domains of pyramidal cells and included (1) O-LM inter-neurons, found to coexpress both SS and parvalbumin (PV); (2) interneurons with target selectivity for other interneurons, expressing vasoactive intestinal polypeptide (VIP) and/or the calcium-binding protein calretinin; (3) procholecystokinin-immunopositive interneurons probably non-basket and dendrite-targeting; and (4) an as-yet unidentified SS-immunoreactive but PV-immunonegative interneuron class, possibly corresponding to oriens-bistratified cells. Estimation of the relative proportion of mGluR1alpha-positive interneurons showed 43%, 46%, and 30% co-labeling with SS, VIP, or PV, respectively. The identification of the specific subclasses of CA1 interneurons expressing mGluR1alpha provides the network basis for assessing the contribution of this receptor to the excitability of the hippocampus.     

Colocalization of metabotropic glutamate receptors in rat dorsal root ganglion cells

Glutamate is the main excitatory transmitter in both central and peripheral nervous systems. Discovery of metabotropic glutamate receptors (mGluRs) made it clear that glutamate can have excitatory or inhibitory effects on neuronal function, with group I mGluRs enhancing cell excitability but group II and III mGluRs decreasing excitability. The present study investigated the colocalization of mGluR subtypes representing groups I, II, or III in rat L5 dorsal root ganglion (DRG) cells. The analyses show that group III has the highest expression, with 75.0% of DRG cells expressing mGluR8, followed by group II, with 51.6% expressing mGluR2/3, followed by group I, with only 6.8% expressing mGluR1alpha. mGluR8 is expressed by small, medium, and large diameter cells. In contrast, mGluR1alpha and mGluR2/3 are expressed by mainly small and medium cells. Approximately half of cells expressing group I mGluR1alpha also express either group II mGluR2/3 or group III mGluR8. These mGluR1alpha double-labeled populations are not likely to overlap since >1.0% of mGluR1alpha are triple-labeled. As expected from the high percentage of single-labeled mGluR2/3 and mGluR8 cells, there is a considerable population of double-labeled cells with approximately 30% of each population expressing both receptors. Due to the fact that the number of mGluR1alpha-expressing cells in the DRG is low, the percentage of triple-labeled cells is also low ( approximately 1-2%). The prevalence of groups II and III indicate that glutamate could have a substantial inhibitory effect of primary afferent function, reducing and/or fine-tuning sensory input before transmission to the spinal cord. These anatomical data highlight the potential inhibitory role glutamate may play in peripheral sensory transmission.     

Modulation of intracellular calcium mobilization and GABAergic currents through subtype-specific metabotropic glutamate receptors in neonatal rat hippocampus

Group I metabotropic glutamate receptors (mGluRs) are coupled to phosphoinositide hydrolysis, and are thought to modulate neuronal excitability, by mobilizing intracellular Ca²⁺. Difference in Ca²⁺ mobilization among subclasses of the receptors has been reported, and regarded as a possible cause of variant neuronal modifications. In hippocampal interneurons, several subclasses of mGluRs including mGluR1 and mGluR5 have been immunohistochemically identified. The subclass-specific physiological effects of mGluRs on neuronal transmission in hippocampus, however, have not been fully elucidated. In the present study, effects of group I mGluR agonist, (S)-3,5-dihydroxyphenylglycine (DHPG) on intracellular calcium concentration were examined in hippocampal interneurons. Application of DHPG increased fluorescence ratio in neonatal CA3 stratum oriens/alveus interneurons. The DHPG-induced calcium mobilization was markedly inhibited by mGluR1-specific antagonist, cyclopropan[b]chromen-1a-carboxylate (CPCCOEt). Inhibition of the calcium elevation by mGluR5-specific antagonist, 6-methyl-2-(phenylazo)-3-pyrindol (MPEP), was weaker than that of CPCCOEt. The fluorescence ratio was not significantly changed by application of mGluR5-specific agonist, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG). DHPG induced calcium responses in CA1 interneurons as in CA3, and the responses were partially inhibited by MPEP treatment. Effects of group I mGluR agonist and antagonist were also investigated, on GABA_A receptor-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) in CA3 pyramidal neurons. The GABAergic sIPSCs were facilitated by DHPG perfusion, and the potentiation was reduced by CPCCOEt, and less distinctly by MPEP. The sIPSCs were not significantly potentiated by CHPG application. These results indicate that mGluR1 is functional in hippocampal interneurons, and DHPG exerts its effect mainly through this receptor at early developmental period.     

Heterogeneity of glycinergic and gabaergic interneurons in the granule cell layer of mouse cerebellum

Interneurons of the cerebellum granule cell layer (GCL) form distinct populations. Golgi cells extend dendrites in the molecular layer (ML) and innervate granule cells. In contrast, Lugaro cells have dendrites confined to the GCL but innervate interneurons in the ML, and globular cells have both their dendrites and axons in the ML. The latter cells were described recently and remain poorly characterized. Although several neurochemical markers have been associated selectively with GCL interneurons, it is unclear how they relate to their morphological classification and neurochemical phenotype (glycinergic and/or gamma-aminobutyric acid [GABA]ergic). Here, we performed a detailed characterization of GCL interneurons in mice expressing enhanced green fluorescent protein (GFP) in glycinergic and GABAergic neurons, respectively. By using immunofluorescence for metabotropic glutamate receptor 2 (mGluR2) and neurogranin as markers, we demonstrate the existence of five non-overlapping subsets of Golgi cells: about 65% are glycinergic/GABAergic and co-express both markers. Two small subsets (5-10%) also contain both neurotransmitters but express only mGluR2; they are distinguished by cell body size and location in the GCL. The fourth subset (15%) is GABAergic only and expresses neurogranin. The fifth subset (5%) is glycinergic only and lacks both markers. Thus, the heterogeneity of Golgi cells suggests that they belong to specific functional circuits and are differentially regulated by mGluRs and Ca(2+)-calmodulin-dependent signaling pathways. In contrast to Golgi cells, Lugaro and globular cells are glycinergic/GABAergic and lack mGluR2 and neurogranin. They each represent at least 15% of GCL interneurons and extensively innervate stellate and basket cells, but not Purkinje cells, emphasizing their contribution to inhibitory control of ML interneurons.